The tick Boophilus microplus (Canestrini, 1887), belonging to the Ixodoidae family, is the main bovine ectoparasite in Brazil and in all tropical and subtropical countries. This parasite is extremely well adapted to the climate of a large part of the country and, coupled with the presence of its hosts distributed over more than 80% of the nation's territory, constitutes a major problem to cattle raising in Brazil. The associated losses are not limited to the drop in production resulting from the intense hemophagia, but can also be related to other damages such as the inoculation of toxins from the salivary glands, depreciation of the hides, influencing the productive capacity of the animals and, chiefly, the transmission of various microorganisms that cause diseases which seriously affect cattle raising in the nation, such as Babesia bovis and Babesa bigemina, with participation also in the epidemiology of Anaplasma marginale.
The damage to livestock caused by ticks manifests itself by various typical actions, such as direct damage by the intense hematophagia originating mainly from the female ticks, and which can be as much as 0.6 to 3 ml per adult female. This results basically in loss of production, and various trials have been conducted to assess this situation. In 1987 (HOLROYD et al., Australian Journal of Experimental Agriculture 28: 1:10) observed that animals which had not been touched by ticks had gained on average 17 kg over a period of three years, as compared to those animals exposed to the parasite. In Brazil, (BRANCO et al 1987, Coletânea de Pesquisas EMBRAPA/CNPO, p. 229-234), found an average weight gain of 34.5 kg in Hereford cattle. In the country, FURLONG 1996 observed a reduced milk production in successive increasing infestations. JONSSON et al (Veterinary Parasitology 78: 66-77, 1998) have estimated that each adult female would be responsible for a drop of 8.9 ml in daily milk production and 1.0 g in loss of weight.
The loss caused by the tick bite and the consequent local inflammation adversely affect the value of the hide, which is of great concern in traditional areas of leather production.
The inoculation of toxins when sucking the blood, natural components of the tick saliva such as prostaglandin inhibitors and other modulators of the inflammatory response, which can lead to a hypersensibility reaction, paralysis of the members and even to testicular hypotrophy.
As to the transmission of pathogenic agents, the important ones in Brazil are the protozoa Babesia bovis and Babesia bigemina, with participation also in the epidemiology of Anaplasma marginale (PATARROYO, 1994, Revista de Patologia Tropical 23:145-146).
One must also include as losses to Brazilian livestock industry the costs of direct control of ticks and of the diseases for which they are carriers. HORN and ATECHE 1985 (A Hora Veterinária 4:12-32) have estimated the direct and indirect losses at 800 million dollars. The Ministry of Agriculture, in a paper covering the two years 1983/1984, raises that amount to one billion dollars per year, of which 40% represent losses in milk production. A paper by HORN in 1988 (Programa nacional de controle de parasitoses, in Curso de Parasitologia Animal 2:21-42) mentions expenditures of US$ 13,800,000.00 with acaricides, which would represent 15% of the nation's total cost of protection.
A number of approaches are used at present for tick control and the treatment of bovines with acaricides, which are chemical substances that kill the ticks. This methodology of control is disadvantageous in that a resistance is built against the chemicals in various tick populations, which necessarily leads to the introduction of new chemical substances for the control; the frequency of chemical treatments in an attempt at effective control of ticks in cattle herds; the harm that such chemical substances can cause to animals, the human health and the environment. Another alternative are the crosses of Bos taurus taurus and Bos taurus indicus, but these hybrids have a lower productive capacity than pure breeds, specifically in the case of dairy cattle, the use of chemical substances being necessary for the control stopping short of building tick resistance in such crossings. Still another proposed alternative for control is the use of pastures which would function in the non-parasitic phase of the parasite. Pasture rotation is also seen as an option since, depending on the resting time of each portion of the pasture, the born larvae would consume their food reserves and die before installing themselves in a host. The use of natural tick predators such as long-legged birds and birds of prey has also been researched, but the biologic and food chain risks of introducing such animals are not yet known. The ant Pheidole megacephala has been the subject of research as an alternative of less ecological impact. Natural parasites such as some bacteries and fungi of the species Beauvenia bassiana and Metarhizium anisopliae have also been analyzed. Another attempt at control has been the use of interspecific crossings of tick species to generate infertile males, but the frequency of intraspecific crossings exceeded that of interspecific crossings. All of the systems mentioned have problems of a practical nature, from the economic and/or ecological standpoints, which prevents their use in most of the geographical cattle raising areas.
In contrast to the foregoing description, the possibility of use of an effective vaccine for tick control constitutes an attractive alternative as compared to the methods available and in use at present.
Various attempts have been made to obtain immunization against the infestation of ticks in bovines. For the purpose of identifying the proteinaceous component and the organ in which the protective immunogenic factor would be found, larvae extracts, nervous tissue, hemocytes, hemolymph, genital tract, and eggs of Boophilus microplus were used, but these attempts were unsuccessful (DAVIDSON, S. 1985, Rural Research 128: 4-8; OPDEBEECK et al., 1988, Immunology 63: 363-367).
In 1989, (WILLADSEN et al., Journal of Immunology 143: 1346-1351) finally isolated and purified a protein which mimicked the alterations found with the gross tick extracts. That glycoprotein was designated Bm86, with an estimated weight of 89 kDa and a pl of 5.5. Subsequently, the amino acid sequence of this protein was determined, and it was found that the sequence was composed of 650 amino acids, of which 10% were cystein, showing a great analogy with the growth factor of the human epidermis (RAND et al, 1989, Proceedings of the National Academic Sciences USA 86:9657-9661).
By means of immunohistochemical techniques and monoclonal panels, the location and distribution of Bm86 in the tick's intestine were determined, and were found in the microvillosities of the membrane of the intestine epithelial cells and highly concentrated near the basal membrane (GOUGH and KEMP, 1933, Journal of Parasitology 79:900-907; LEE and OPDEBEECK, 1994, International Journal of Parasitology 25: 241-248).
Trials of vaccination with this protein reportedly show that the antibodies produced in bovines against the protein, as well as those generated by the inoculation of tick intestine extracts, are aggressive to the intestinal epithelium of the parasite, causing irreparable damage which will later affect the biological cycle of this ectoparasite.
Genetic studies which propitiated large-scale production of the protein through DNA cloning which encodes the Bm86 as Escherichia coli (TELLAM et al, 1992, Animal Parasite Control Using Biotechnology: 303-331), as Aspergilus nidulans and as Baculovirus (TURNBULL et al, 1990, Applied Environmental Microbiology 56: 2847-2852; TELAMM et al. 1992), have been performed, as well as vaccination trials, with up to 70% protection in some cases, resulting in a decrease of tick growth and affecting its biological cycle. Then, in 1994 the first vaccine against tick, named TICKGARD® was commercially launched, through genetic engineering techniques in E. coli (SMITH et al, 1995).
In 1994 the second commercial vaccine against Boophilus Microplus was launched under the trade name GAVAC®, it was developed by Cuban research groups who expressed Bm86 in the yeast Pichia pastoris (RODRIGUEZ et al, Journal of Biotechnology 33: 135-146, 1994).
The third vaccine would come in 1996, the TICKGARD PLUS®, which has been successfully tested in various bovine breeds (WILLADSEN et al, Veterinary Parasitology 71: 209-222. 1997).
Notwithstanding the foregoing description, where it is shown that the use of commercial immunogens can result in significant reduction of infestation, they do not yet offer levels of protection which would make the use of conventional acaricides.
The purification procedures and obtention of immunogens for ticks in natura require an extremely ample and complex procedure. On the other hand, obtaining recombinant proteins requires the inconvenient preparation and handling of extensive cell cultures, either the fermentation or others intended for getting the proteinaceous material necessary for mass immunization, which is costly and may involve highly complex technical problems.
Obtaining new immunogens which can function as alternatives and/or complements to those already described represents a considerable advance in the development of effective vaccines.
In this context, we can note the propensity for new research, prompted by the recent progress of the knowledge in the areas of immunology, biochemistry, cell biology and biotechnology, for control of these parasites by vaccination.
Taking into account the fact that a protein can have several epitopes in its structure, which can act as protecting factors or not, and which can even be a mechanism of parasite mimicking to evade the immune response, it would be greatly advantageous to utilize vaccines having determinate and characterized epitopes, since this would avoid some problems brought about by the epitopes which are not necessary for developing protection and immunity against the parasite, such as suppressing, allergic and/or self-immune mechanisms, and typical evasion mechanisms of microorganisms.
Thus, a tick control vaccine based on the chemical synthesis of an immunogen would be more advantageous than a recombinant vaccine, such as high purity, because it does not require costly and complex purification techniques, its complete chemical characterization, it is safe because of the absence of contaminants, complete large-scale reproducibility, high stability, since it does not contain enzymes and other proteinaceous materials derived from other biologic materials, making its storage easier, and lower cost of production on an industrial scale.